Compound for treating oxidative stress in mammals

ABSTRACT

Managing and treating elevated OS biomarkers in mammals such as companion animals with at least one of the supplements alpha-lipoic acid, carnitine, co-enzyme Q-10, ginger, green tea, licorice, milk thistle, garlic, honey. resveratrol, soybeans, tomatoes, turmeric, vitamin D, vitamin E or selenium. Diagnosing an oxidative stress (OS) in a mammal comprises collecting a sample; screening the sample to detect the presence of an OS biomarker, selectively isoprostane and other antioxidants such as HODE microRNAs. TAC: GSH, MDA, and TNF-alpha. The sample can be saliva.

RELATED APPLICATION

This application relates to U.S. Provisional Patent App. No. 62/953,049for OXIDATIVE STRESS BIOMARKERS TESTING IN ANIMALS filed Dec. 23, 2019;Attorney Docket 058034-020000. The contents of this application areincorporated by reference in its entirety herein, and priority isclaimed from that application. This application also relates to U.S.patent application Ser. No. 17/114,156 for OXIDATIVE STRESS BIOMARKERSTESTING IN ANIMALS by W. Jean Dodds and Denis M. Callewaert filedcontemporaneously with the present application on Dec. 7, 2020; AttorneyDocket 058034-020001. The contents of those applications areincorporated by reference in its entirety herein.

BACKGROUND

The present disclosure is directed to cellular oxidative stresstreatment in mammals such as companion animals.

In people and animals, cells in homeostatic equilibrium are in a stateof oxidative balance, namely, when there are sufficient cellularantioxidants (both small molecule radical scavengers and enzymes such ascatalase and superoxide dismutase) to avoid a buildup of excess freeradicals (oxidizing agents). However, when levels of oxidizing agentsexceed antioxidants, the condition is termed oxidative stress (OS). OSis now known to trigger inflammatory responses and to be an underlyingcause of most chronic diseases.

Extensive investigations of OS and chronic tissue inflammatory responseshave established that they are major underlying risk factors that playkey roles in the etiology of a range of human (and animal) diseases,including rheumatoid arthritis, cancers, diabetes, obesity, periodontaldisease, neurodegenerative disorders, and cardiovascular diseases. Thedevelopment and progression of these chronic diseases is influenced by arange of environmental, dietary, and lifestyle factors, and specificdietary components, that—along with exercise and some nutraceuticals—cansignificantly impact OS and inflammation.

Chronic inflammation resulting from increased free radical formation inOS, also called reactive oxygen species (ROS), occurs when tissues ororgans receive inflammatory “mediator” messages that cause them to reactas though a “trigger” such as a pathogen is present. Rather thanrepairing themselves, these cells can remain in an ongoing state ofinflammation that can wax and wane for prolonged periods. Tissues thusbecome deficient in antioxidant mediators, such as glutathione,cysteine, ascorbic acid and other radical scavenging vitamins, as wellas superoxide dismutase, catalase and other antioxidant enzymes, andthese deficiencies are strongly correlated with poor clinical outcomes.For mammals in good health, ˜25% of oxygen intake gives rise to ROS,whereas this increases with age and poor health to as much as 75%.

When cells undergo damage due to OS and inflammation, the incidence andseverity of infections, obesity cardiovascular disease and cancersincrease. These damaged cells release molecules that can be used asbiomarkers for OS and inflammation. Biomarkers of clinical relevance inpeople and pets include isoprostanes, malondialdehyde, and severalcytokines including tumor necrosis factor (TNF), hydroxyoctadecadienoicacid (NODE), as well as certain microRNAs. Elevated levels of thesebiomarkers in biofluids (including plasma, urine and/or saliva) can beused to evaluate oxidative status and can addressed with diet andsupplement changes to promote beneficial antioxidant effects. On theother hand, alternative biomarkers, such as glutathione, certainmicroRNAs, and some enzymes are indicative of a healthy antioxidantcondition. Biomarkers can be analyzed assays periodically to assess theresponse and adjust lifestyle, nutritional and nutraceutical therapy, asneeded.

Significant efforts have been made toward the dietary management of OS.In contrast to small molecule antioxidants (e.g., ascorbic acid) thatcan neutralize one ROS/molecule, recent studies have focused on thedevelopment of functional foods, e.g., those containing natural Nrf-2activators since Nrf-2 upregulates expression of multiple antioxidantenzymes including superoxide dismutase, catalase, glutathionetransferases and glutathione reductase. A single molecule of one ofthese enzymes can neutralize a huge number of ROS. Significant amountsof antioxidant radical scavengers and/or Hrf-2 activators areingredients in many functional foods and supplements such as: turmeric(Curcuma longa); and its relative, ginger (Zingiber officiale); chilipeppers (Capsicum annuum); green tea (Camellia sinensis, which containstannins and polyphenol catechins, and other teas); soybeans (Glycinemax); tomatoes (Solanum lycopersicum, rich in lycopenes); grapes (notfor pets); honey (not for infants or very young animals); cranberries(Vaccinium macrocarpon, contains pro-anthocyanidins); licorice(Glycyrrhiza glabra); garlic (Allium sativum, in moderation for pets);milk thistle (Silybum marianum);cabbages and broccoli.

Some Primary Oxidative Stress Biomarkers

Isoprostanes are a series of prostaglandin-like compounds produced bynon-enzymatic ROS-catalyzed peroxidation of arachidonic acid. Theconcentration of isoprostanes is considered the “gold standard” forquantifying OS in vivo in humans and certain animals.

MicroRNAs are small non-coding RNA molecules found mostly in the cellsof plants, animals and some viruses. They function in RNA silencing andpost-transcriptional regulation of gene expression. The function ofmicroRNAs is in gene regulation.

Reduced glutathione (GSH) plays two key roles in the maintenance ofredox balance. First, GSH is a major intracellular radical scavengerthat can react with and neutralize ROS thus preventing ROS-mediateddamage to macromolecules such as proteins and DNA.

GSH is also a co-substrate for Glutathione-S-Transferases (GSTs), asuperfamily of isozymes best known for their ability to catalyze theconjugation of the reduced form of glutathione (GSH) to foreignsubstrates for the purpose of detoxification. GSTs are found in plants,animals, fungi, and some bacteria. In vertebrates there are over 18distinct cytosolic GSTs which are expressed most tissues, but especiallyin liver, kidney, heart, lung, and brain tissues. High levels of GST areassociated with resistance to the apoptosis (cell death) induced by arange of substances, including chemotherapeutic agents. The levels ofspecific GST isoforms in urine and serum are indicators of hepatocyteand renal tubular injury in transplantation, toxicity and viralinfections of humans and rodents.

The production of GSH is dependent on the enzyme glutathione reductase(GSR), and the expression of GSR, as well as multiple cytosolic GSTs andother antioxidant enzymes including superoxide dismutase are upregulatedby the transcription factor Nrf-2. Therefore, while direct measurementof Nrf-2 activation by diet, nutraceuticals, etc. is extremelydifficult, the levels of glutathione in biological fluids can serve as asurrogate biomarker for Nrf-2 activation.

Malondialdehyde (MDA) is a key byproduct of the peroxidation ofpolyunsaturated fatty acids. It is very reactive and forms adducts withmacromolecules, thereby altering or eliminating their function. MDAand/or MDA adducts are well established biomarkers for OS. However, somemethods for the measurement of MDA, including the thiobarbituric acidreactive substance (TBARS) assay as it was traditionally performed donot provide consistent reliable results. Malondialdehyde is potentiallymutagenic, and has been found in heated edible oils such as sunflowerand palm oils. Corneas of human patients suffering from keratoconus andbullous keratopathy have increased levels of malondialdehyde, and thisaldehyde also can be found in tissue sections of joints from humanpatients with osteoarthritis.

Tumor Necrosis Factor-Alpha (TNF-α), also called cachexin or cachectin,is a cell signaling protein involved in systemic inflammation and is oneof the cytokines that play a primary role in the acute phaseinflammatory response. It is produced chiefly by activated macrophages,although it can be produced by many other cell types such as CD4+lymphocytes, natural killer (NK) cells, neutrophils, mast cells,eosinophils, and even neurons.

TNF is an endogenous pyrogen that can induce fever, apoptotic celldeath, cachexia, inflammation, inhibit tumorigenesis and viralreplication, and respond to sepsis via interleukin IL-1 and IL-6producing cells. Dysregulation of TNF production has been implicated ina variety of human diseases such as Alzheimer's disease, cancer,depression, psoriasis and inflammatory bowel disease (IBD).

Hydroxyoctadecadienoic Acid (HODE) is a lipid peroxidase biomarker foundto be elevated for early detection of periodontal disease, type-2diabetes, rheumatoid arthritis, cataracts, Alzheimer's disease andhepatitis-B and -C in people.

Other Biomarker Enzymes include: Sorbitol Dehydrogenase, a cytosolicenzyme that converts sorbitol, the sugar alcohol form of glucose, intofructose; and 5′ Nucleotidase, which catalyzes the phosphorolyticcleavage of 5′ nucleotides, and is considered a maturation marker forT-and B-cells.

Measuring Biomarkers of Oxidative Stress

Traditionally, cellular biomarkers are measured in the serum and/orurine from humans and animals. However, collecting these samplesespecially from children and smaller animal species presents withdifficulty and causes unnecessary stress.

Neither canine whole blood-, serum-, nor urine-based isoprostanequantitations are accurate, linear and predictive as a marker for tissueoxidative stress in the canine species. Similarly, there are nopublished studies of measuring HODE as a biomarker of OS in companionanimals

There is a need for another form of OS testing in companion animals,especially canines.

Managing and Treating Elevated Biomarkers of Oxidative Stress. Use oftreatments and preventive health measures including applicable drugs,diets, supplements and exercise.

SUMMARY

The disclosure includes management and treatment measures to alleviateelevated oxidative stress biomarkers and their clinical expression incompanion animals such as dogs or cats, and other animal species.

According to the disclosure, measurement is affected in bodily fluids,selectively saliva.

Collection of saliva is noninvasive, painless, relatively inexpensiveand convenient for the individual. Salivary testing of OS and/orinflammatory biomarkers can reveal the latent or pre-clinical form ofdeveloping OS.

There is a need to provide for practical and rapid screening or testingfor OS to permit enhancement of the health of animals. Current methodsand findings in humans and rodents measure OS biomarkers primarily inserum or urine. However, while one can measure OS biomarkers in healthydog serum, neither serum nor urine has provided reliable quantitativeand linear measurements in healthy dogs or especially in those withchronic diseases, where one would predict their elevation from tissueOS.

In accordance with this disclosure there is provided a diagnostic testsystem for OS stress assessment in animals, in particular companionanimals, such as dogs, cats, rabbits, hamsters, and horses.

Saliva can be used as a diagnostic tool to assess the health or diseasestatus of an animal. Saliva is easily collected, stored and shipped, andprovides a non-invasive means of multiple or serial sampling for use asa diagnostic tool for a variety of conditions in animals.

The disclosure uses a species-specific test for companion animals suchas dogs or cats, and other animal species, and the appropriate methods.

The disclosure is further described in detail.

DETAILED DESCRIPTION

Different biomarkers are described and different abbreviations below areas follows.

IsoP, isoprostane(s); AOX, antioxidant capacity; CRP, C-reactiveprotein; GSH, glutathione; MDA, malonaldehyde; NO, nitric oxide; TAC,total antioxidant capacity; transcription factor Nrf-2 (Nuclear factorerythroid 2-related factor 2), TNF-alpha, tumor necrosis factor-alpha,and HODE, hydroxyoctadecadienoic acid.

The disclosure provides a straightforward, reliable assay for OSbiomarkers and for antioxidant capacity of biological fluids.

The present disclosure relates to a test for levels of oxidized lipidbiomarkers, selectively isoprostanes, HODE, microRNA and otherbiomarkers for animals, particularly companion animals and moreparticularly dogs, cats or horses using saliva that permits the rapid,accurate, non-invasive quantitative screening for biolipids, selectivelyin the animal.

The saliva-based test assay quantifies the isoprostane, HODE andmicroDNA levels in dog saliva to determine if the pet's body isundergoing harmful OS. OS creates reactive oxygen species (ROS) causingcells to undergo damage and release biomarker lipids and enzymes thatlead to tissue inflammation, infections, periodontal disease, obesityand even cancers. However, free radicals themselves are so reactive andshort-lived that direct measurement is not possible. Thus, isoprostaneand HODE levels serve as reliable surrogate biomarkers for the presenceof ROS.

The saliva-based tests of this disclosure are novel isoprostane, HODEand microRNA tests, and are examples of a set of unique biomarker testsfor pets that can be measured in saliva.

For quantitative testing, an animal's serum or saliva or other bodilyfluid is added to the ELISA microtiter plate or other immunoassayplatforms such as but not limited to lateral flow, or latex or beadagglutination, which measures the presence of cellular oxidative stress.

Once collected at or received by the lab, the blood serum or saliva orother bodily fluid sample is then screened using the ELISA method orother immunoassay platforms such as but not limited to lateral flow, orlatex or bead agglutination, which measures the presence of OS.

Forms of biological fluid, other than saliva, for instance urine, tears,sweat, or milk or other mucosal secretions can be used.

The detection of cellular oxidative stress can be performed with animmunoassay. Immunoassays include, but are not limited to, ELISA test,RIA test, latex agglutination, beads assay, and proteomic assays. Apreferable immunoassay is the ELISA test. Other immunoassays can be usedand the choice of immunoassay can be determined by one of ordinary skillin the art.

A method for diagnosing OS in a mammal such as a human, dog or othercompanion animal comprises the steps of collecting a sample of saliva;screening the sample to detect a level of at least one of a number of OSmarkers and detecting and diagnosing the presence of OS based on thelevel of one or more markers.

A method for diagnosing OS in a mammal such as human, dog or othercompanion animal comprises the steps of collecting a sample of saliva;screening the sample to detect a level of at least one isoprostane, anddetecting and diagnosing the presence of OS based on the quantitativelevel of the isoprostane.

A method for diagnosing OS in a mammal such as human, dog or othercompanion animal comprises the steps of collecting a sample of saliva;screening the sample to detect a level of at least one HODE, anddetecting and diagnosing the presence of OS based on the quantitativelevel of the HODE.

The method further comprises collecting a first testing portion of thesaliva sample and wherein the first testing portion is the sample foruse in the screening step. [Glucuronidase pretreatment is not requiredfor saliva samples.]

The method includes the screening step utilizes an enzyme-linkedimmunosorbent assay (ELISA) testing system to detect the level of thesaliva-based OS marker.

Results

After completing the initial clinical trial studies, analyzing 282clinical patient samples; 79 of them (35%) were positive, havingisoprostane biolipid levels, for example, above the normal referencerange we have established (i. e., 0.5-1.75 ng/mL of saliva).

Of the 79 positive testing dogs, there were: 34% spayed females, 32%neutered males, 21% intact males and 15% intact females. The ages rangedfrom 4 months to 15 years, although most were middle aged or older. Theweight range was 4-143 pounds, with 84% being medium to large or giantin size; no breed type predominated.

The diets fed the 79 positive dogs included: 40% ate only a commercialraw diet; 20% only a commercial dry kibble; 13% a home cooked or homeprepared raw diet, and 3% ate a combination of a commercial kibble andraw.

Of the 79 positive dogs, 38 also had saliva-based profiles run for foodsensitivity and intolerances to 24 foods. Interestingly, only 3 of these38 dogs had Saliva based test results that were reactive (20 or morefoods). These results suggest that dogs with clinical issues related tointense itching, scratching, chewing, and bowel irritability hadrelatively few identified foods as the culprits. Environmental exposureto inhalants, fleas, ticks, mites and other insects as well as contactreactants could be involved; and 3 dogs were taking an isooxazolineparasiticide.

Positive testing dogs should be retested in about 6 months after beingon foods and supplements designed to lower their OS. The beneficialoutcome of 50 isoprostane positive samples revealed reduced isoprostanelevels when retested 5-6 months after taking the supplements listedbelow.

Management and Treatment

Management and treatments measures to alleviate elevated oxidativestress biomarkers of at least isoprostane and/or HODE, and theirclinical expression in companion animals such as dogs or cats, and otheranimal species are described below. Use of supplements for isoprostaneand HODE can be used for any other biomarkers of OS such as MicroRNAsPGF2a, MDA, TAC and others. Supplements that are used to bring down highlevels of isoprostane and/or HODE are:

-   Alpha-Lipoic acid-   Carnitine-   Co-Enzyme Q-10-   Ginger-   Green tea-   Licorice-   Milk thistle, and a few more like garlic and honey.-   Resveratrol (as a natural supplement or as food like blueberries and    cranberries)-   Selenium-   Soybeans-   Tomatoes-   Turmeric (curcumin)—without black pepper for pets-   Vitamin D-   Vitamin E

A synergistic biological effect of mutual benefit, based upon theprinciple of cooperative symbiosis, is achieved by this group ofingredients.

Two or more of Alpha-Lipoic acid; Carnitine, Co-Enzyme Q-10, Ginger,Green tea, Licorice, and Milk thistle produce enhanced and thus aresynergistic.

The synergy results from the interaction between nutrients, theirabsorption and bioavailability in the body which in this situation ispositive and provides health benefits. Evidence-based studies haveillustrated that the active components in the nutritional supplements ofthe disclosed supplements work together synergistically to enhance theirfunctional properties in preventing diseases and betterment of health.The role of the disclosed nutritional supplements working togetherenhances treatment and prevention of chronic diseases, such ascardiovascular and periodontal disease, infections, dysbiosis, diabetes,obesity, and cancers. This combination synergic effect is a scientificadvance in nutritional therapy and significantly brings down high levelsof isoprostane and/or HODE more effectively than when the supplementsare separately used.

Additional synergistic effects can be obtained with Garlic and honey,Resveratrol (as a natural supplement or as food like blueberries andcranberries); Selenium; Soybeans; Tomatoes; Turmeric (curcumin); VitaminD; and Vitamin E.

Each of these individually produce overtly similar effects, they displaygreatly enhanced effects when given in combination. The combined effectis greater than that predicted by their individual potencies, and thusthe combination is synergistic.

The compound of the disclosure includes treating an elevated isoprostaneOS biomarker with at least one of alpha-lipoic acid, co-enzyme Q-10,green tea, turmeric, vitamin D, or selenium.

The method of the disclosure includes treating an elevated isoprostaneOS biomarker and an elevated HODE OS biomarker with at least one ofalpha-lipoic acid, co-enzyme Q-10, green tea, turmeric, vitamin D, orselenium.

The compound of the disclosure includes treating an elevated HODE OSbiomarker with at least one of alpha-lipoic acid, co-enzyme Q-10, greentea, turmeric, vitamin D, or selenium.

The compound of the disclosure includes treating an elevated OSbiomarker being at least TAC, GSH, MDA, TNF-alpha, NO, and microRNA withat least one of alpha-lipoic acid, co-enzyme Q-10, green tea, turmeric,vitamin D, or selenium.

The compound of the disclosure includes treating an elevated isoprostaneOS biomarker with at least alpha-lipoic acid, co-enzyme Q-10, green tea,milk thistle, garlic, honey. resveratrol, soybeans, tomatoes, turmeric,vitamin D, or selenium.

The compound of the disclosure includes treating an elevated HODE OSbiomarker with at least alpha-lipoic acid, co-enzyme Q-10, green tea,milk thistle, garlic, honey. resveratrol, soybeans, tomatoes, turmeric,vitamin D, or selenium.

The compound of the disclosure includes treating an elevated isoprostaneOS biomarker with at least one of, or with at least two of, or with atleast three of, or with at least four of, or with at least five of, orwith at least six of, or with at least seven of, or with at least eightof, or with at least nine of, or with at least ten of alpha-lipoic acid,carnitine, co-enzyme Q-10, ginger, green tea, licorice, milk thistle,garlic, honey. resveratrol, soybeans, tomatoes, turmeric, vitamin D,vitamin E or selenium.

The compound of the disclosure includes treating an elevated isoprostaneOS biomarker and an elevated HODE OS biomarker with at least one of, orwith at least two of, or with at least three of, or with at least fourof, or with at least five of, or with at least six of, or with at leastseven of, or with at least eight of, or with at least nine of, or withat least ten of: alpha-lipoic acid, carnitine, co-enzyme Q-10, ginger,green tea, licorice, milk thistle, garlic, honey. resveratrol, soybeans,tomatoes, turmeric, vitamin D, vitamin E or selenium.

The compound of the disclosure includes treating an elevated HODE OSbiomarker with at least one of, or with at least two of, or with atleast three of, or with at least four of, or with at least five of, orwith at least six of, or with at least seven of, or with at least eightof, or with at least nine of, or with at least ten of alpha-lipoic acid,carnitine, co-enzyme Q-10, ginger, green tea, licorice, milk thistle,garlic, honey. resveratrol, soybeans, tomatoes, turmeric, vitamin D,vitamin E or selenium.

The compound of the disclosure includes treating an elevated OSbiomarker being at least TAC, GSH, MDA, TNF-alpha, NO, and microRNA withat least one of, or with at least two of, or with at least three of, orwith at least four of, or with at least five of, or with at least sixof, or with at least seven of, or with at least eight of, or with atleast nine of, or with at least ten of alpha-lipoic acid, carnitine,co-enzyme Q-10, ginger, green tea, licorice, milk thistle, garlic,honey. resveratrol, soybeans, tomatoes, turmeric, vitamin D, vitamin Eor selenium.

The compound of the disclosure includes treating an elevated OSbiomarker being at least TAC, GSH, MDA, TNF-alpha, NO, and microRNA withat least one of, or with at least two of, or with at least three of, orwith at least four of, or with at least five of, or with at least sixof, or with at least seven of, or with at least eight of, or with atleast nine of, or with at least ten of: alpha-lipoic acid, carnitine,co-enzyme Q-10, ginger, green tea, licorice, milk thistle, garlic,honey. resveratrol, soybeans, tomatoes, turmeric, vitamin D, vitamin Eor selenium.

The proportional decrease in the biomarker level of the oxidativestress, being the value of the isoprostane and/or HODE has been at least40% and in some cases as much as 90% after about 5-6 months of ingestionof the compounds of the disclosure.

The compound is formulated with the ingredients being mixed together inpowdered form and being for ingestion and digestion by the mammal,selectively an animal at least once or twice per day together with thefood that is being eaten. In this format, the powder is mixed into thefood prior to the food being given to the mammal such as human oranimal.

General

Many different formats are possible. In the specification, there havebeen disclosed typical embodiments of the disclosure. Although specificterms are employed, they are used in a generic and descriptive senseonly and not for purposes of limitation, the scope of the disclosurebeing set out in the claims. It is therefore to be understood thatwithin the scope of the appended claims the disclosure may be practicedotherwise than as described.

1-20. (canceled)
 21. A veterinary nutritional compound for treating anelevated isoprostane OS biomarker in a dog with supplements of at leastvitamin D and selenium, wherein the isoprostane biomarker in the dog sotreated is reduced by at least 40% after five to six months from a levelbelow 1.75 ng/ml as measured in saliva from the dog.
 22. A veterinarynutritional compound as claimed in claim 21 including the supplements ofat least alpha-lipoic acid, co-enzyme Q-10, green tea, turmeric.
 23. Aveterinary nutritional compound as claimed in claim 21 includingtreating an elevated HODE OS biomarker in the dog.
 24. A veterinarynutritional compound as claimed in claim 22 including treating anelevated HODE OS biomarker in the dog.
 25. A compound as claimed inclaim 21 including at least one of supplements selected from the groupconsisting of alpha-lipoic acid, co-enzyme Q-10, green tea, turmeric.26. A compound as claimed in claim 21 including at least two supplementsselected from the group consisting of alpha-lipoic acid, co-enzyme Q-10,green tea, turmeric.
 27. A compound as claimed in claim 21 including atleast three supplements selected from the group consisting ofalpha-lipoic acid, co-enzyme Q-10, green tea, turmeric.
 28. A compoundas claimed in claim 21 including at least two supplements selected fromthe group consisting of carnitine, ginger, licorice, milk thistle,garlic, honey. resveratrol, soybeans, tomatoes, turmeric, and vitamin E.29. A compound as claimed in claim 21 including at least threesupplements selected from the group consisting of carnitine, ginger,licorice, milk thistle, garlic, honey. resveratrol, soybeans, tomatoes,turmeric, and vitamin E.
 30. A compound as claimed in claim 22 includingat least two supplements selected from the group consisting ofcarnitine, ginger, licorice, milk thistle, garlic, honey. resveratrol,soybeans, tomatoes, turmeric, and vitamin E.
 31. A compound as claimedin claim 22 including at least three supplements selected from the groupconsisting of carnitine, ginger, licorice, milk thistle, garlic, honey.resveratrol, soybeans, tomatoes, turmeric, and vitamin E.